1. Field of the Invention
The present invention relates generally to assays for detecting lead in samples suspected of containing lead. More particularly, the invention concerns a lead assay capable of performing whole blood lead assays rapidly and easily on commercially available automated assay systems.
2. Background Discussion
The toxicity of lead is well-known. Trace amounts of lead can cause severe damage to human organs. Moreover, given the numerous and widespread sources of lead in the environment, the ability to rapidly detect even very low levels of lead in various human or environmental samples is becoming increasingly important to public health.
It is generally recognized that lead poisoning occurs in children at blood levels as low as 10-15 ug/dl. Lead contamination of environmental sources such as water, dust and soil requires identification at even lower levels. To measure these trace amounts, analytical techniques must be sensitive, contaminant-specific, and reliable Unfortunately, one well-known technique for accurately determining the presence of trace amounts of lead in a sample, a method known as atomic absorption, is both costly and time-consuming. Another well-known technique is anodic stripping voltametry. A drawback with this method is that it performs only one test at a time and usually involves the use of mercury compounds.
Another technique for determining the presence of lead involves the use of enzymes which are capable of being inhibited by lead. Enzymes are complex biological molecules which act as catalysts for various chemical reactions that occur in nature. One such enzyme, aminolevulinic acid dehydratase ("ALAD"), is inhibited in its effectiveness as a biological catalyst when exposed to lead. A known way to utilize this phenomenon to determine the presence of lead in a sample is to combine the sample suspected of containing lead with a mixture of ALAD and another compound known as aminolevulinic acid ("ALA"). ALA is a substrate for ALAD, meaning that ALAD will react with ALA. This reaction produces a reaction product called porphobilinogen ("PBG"). Under normal circumstances, when lead is not interfering with ALAD, the enzyme will convert its substrate ALA into PBG. However, when lead inhibits ALAD's ability to react with ALA, the reaction product PBG will not be formed, or will be formed in lesser amounts.
Thus, when a sample suspected of containing lead is mixed with ALAD and ALA, one can determine the presence of lead by observing whether and to what extent PBG is formed. If PBG production is reduced in a sample suspected of containing lead, this is an indication that lead is present in the sample and inhibiting the reaction between ALAD and ALA. In this enzyme-based test for lead, the presence of PBG is normally detected by using a coloring reagent which reacts with the PBG. After the PBG has been reacted with the coloring reagent, its presence can be determined colorimetrically--i.e. by transmitting light through the sample and measuring how the presence of the colored PBG affects the wavelength of light absorbed by the sample.
There are a number of problems which have prevented the ALAD-based technique discussed above from achieving widespread acceptance as a clinical test for lead. A principal problem is that the ALAD test is often not sensitive enough to detect the very low concentrations of lead in whole blood which are now generally regarded as toxic concentrations. For example, in an ALAD assay in which ALAD and ALA are added to a sample to detect potential inhibition by lead, the assay cannot be performed directly on whole blood. Instead, a blood sample usually is first treated with acid to release the lead from the red blood cells. The sample is then centrifuged to remove proteins and other materials which would interfere with the assay. The supernatant obtained after centrifugation must then be neutralized before ALAD and ALA can be added and incubated therein. Lead present in the blood sample tends to be poorly recovered as a result of these procedures such that it is either lost from the supernatant through precipitation, and the like, or is present in a form which is not readily accessable by the ALAD. Hence, analysis for porphobilinogen after ALAD and ALA are incubated in a whole blood supernatant may falsely indicate that the original whole blood sample does not contain a toxic level of lead. Absent a treatment method for blood samples in which all or essentially all of the lead in a blood sample can be isolated (i.e., recovered) into a supernatant of the blood in a form which can be detected by the ALAD assay, such assays directed to whole blood have serious limitations. To the best of our knowledge, it has heretofore been impossible to perform an ALAD-type assay for lead on a whole blood sample, much less one which is fully or partially automated, in such a manner that the results, in terms of concentration of lead detected, are comparable to the known methods of atomic absorption and anodic stripping voltametry.
Another difficulty associated with assays that use disulfide enzyme inhibition is that the enzyme must be activated, i.e. reduced, with a reducing agent. In known ALAD assays, the reducing agents are typically sulfhydryl compounds such as dithiothreitol. Unfortunately, these compounds interfere with color development of the porphobilinogen reaction product and hence must be precipitated from the assay solution. Known compounds typically used for effecting such precipitation are mercury salts which are unattractive from an environmental and toxicological standpoint. There is a pronounced need for alternatives to mercury salts. Ideally, it would be most desirable if a non mercury-based reducing agent could be discovered which would not need to be precipitated from the assay solution.
Yet another significant drawback in the ALAD assay is that it is labor intensive. To our knowledge, no one has succeeded in automating such an assay. Automation of the ALAD assay, especially for measuring lead concentration in whole blood, would be considered very attractive for clinical laboratories that require fast turnaround with lean staffing. Among the most popular automated analytical instruments currently employed in clinical laboratories worldwide are the IMx.RTM. Analyzer, the AxSYM.RTM. analyzer, the TDx.RTM. analyzer and the Vision.RTM. analyzer, all of which are manufactured by Abbott Laboratories. There exists a need in the art for a highly sensitive and accurate lead assay capable of being performed with little or no manual intervention on these types of computer-driven laboratory analyzers. It would be particularly advantageous to provide an automated enzyme-inhibition assay for lead in which the enzyme/substrate reaction product could be detected using the colorimetric or fluorometric detection systems which are found in commercially available analyzers such as the above-named instruments.